Detailing the Feedback Interplay between Phosphoinositides, Rho GTPases and Actin Governing Cell Polarisation

• Applicant: Dr. Oliver Rocks
• Subject Area: Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 503783151

An important manifestation of cell polarity is the establishment of a front-rear axis in migrating cells. Front polarisation is driven by feedback interactions between the signalling modules phosphoinositide-3 kinase, Rho GTPases and actin. While the key players of front polarity are well studied, we still have little understanding of the molecular mechanisms by which they intersect to give rise to self-organised morphodynamics. RhoGEF and RhoGAP proteins may be key elements in this feedback as they provide context to cytoskeletal signalling and are engaged by phosphoinositide lipids (PIPs) for polarisation. Although two third of the 145 human Rho regulators encode potential PIP binding domains, specific interactions with and regulation by PIPs are unclear. We have previously established tools for family-wide studies of RhoGEFs/RhoGAPs. Our work has revealed that their interaction with actin arrays can account for feedback phenomena that shape Rho signalling patterns. To further explore their role in polarity feedback, we systematically screened for PIP-responsive GEFs/GAPs and found that almost all PIP3-recruited regulators (9/11) associate with actin. We therefore hypothesise for these hits a yet undescribed “triple ‘AND’-gate/feedback” mechanism that couples input from all three front-polarity signalling elements (PIP/Rho/actin) to sustain exploratory cell polarisation. Preliminary work on a Cdc42/Rac GEF supports this mechanism. The main goal of this proposal is to detail this spatially organising feedback circuit and its role in leading-edge polarisation and steering. Secondly, we aim to make use of the modular design of this mechanism to engineer synthetic ‘polariser proteins’, based on an actin binding motif, a GEF and a PH domain, to rewire the feedback and systematically explore parameters that determine polarised cell shape. Prior to this, we will complement our assay tools and refine the mapping of PIP-responsive GEFs/GAPs to comprehensively assess the relevance of PIP2, PIP3 and PI(3,4)P2 in Rho signalling regulation. Understanding the organisation principles that translate polarity protein interactions into morphogenic and migratory cell behaviour is of great fundamental and clinical interest and may reveal strategies for therapeutic interference.

• DFG Programme: Research Grants